PLANT PHYSIOLOGY , Vol 102, Issue 3 761-769, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Catalytic Properties of a Newly Discovered Acyltransferase That Synthesizes N-Acylphosphatidylethanolamine in Cottonseed (Gossypium hirsutum L.) Microsomes
K. D. Chapman and T. S. Moore Jr
Botany Department, Louisiana State University, Baton Rouge, Louisiana 70803
We recently demonstrated that cotyledons of cotton (Gossypium hirsutum L.)
seedlings synthesize N-acylphosphatidylethanolamine (NAPE), an unusual
acylated derivative of phosphatidylethanolamine (PE), during
postgerminative growth (K.D. Chapman and T.S. Moore [1993] Arch Biochem
Biophys 301: 21-33). Here, we report the discovery of an acyltransferase
enzyme, fatty acid: diacylphosphatidylethanolamine N-acyltransferase
(designated NAPE synthase), that synthesizes NAPE from PE and free fatty
acids (FFA) in cottonseed microsomes. [14C]NAPE was synthesized from
[14C]palmitic acid and endogenous PE in a time-, pH-, temperature-, and
protein concentration-dependent manner. [14C]Palmitic acid was incorporated
exclusively into the N-acyl position of NAPE. [14C]palmitoyl coenzyme A
(CoA) and [14C]-dipalmitoyl phosphatidylcholine (PC) were poor acyl donors
for the synthesis of NAPE (i.e. 200- and 3000-fold lower incorporation
efficiency than palmitic acid, respectively). Synthesis of NAPE from
palmitoyl-CoA and dipalmitoyl-PC was observed only after the release of FFA
in microsomes. We observed a temperature optimum of 45[deg]C and a pH
optimum of 8.0 for the synthesis of [14C]NAPE from [14C]palmitic acid (or
from [14C]PE). NAPE synthase activity showed no apparent divalent cation
requirement. Notably, activity was stimulated by HPO42-, HCO3-, SO42-, and
NADPH, whereas activity was inhibited by Ca2+, Mn2+, Cd2+, ATP, ADP, flavin
adenine disnucleotide, and flavin mononucleotide. Other nucleotide
triphosphates (GTP and CTP) and pyridine dinucleotides (NAD, NADH, and
NADP) did not appreciably affect NAPE synthase activity. Initial velocity
measurements of NAPE synthase activity at increasing concentrations of
palmitic acid showed non-Michaelis-Menten, biphasic kinetics. A
high-affinity site (S0.5 = 7.2 [mu]M, Vmax = 18.8 nmol h-1 mg-1 of protein)
and a low-affinity site (S0.5 = 32.0 [mu]M, Vmax = 44.9 nmol h-1 mg-1 of
protein) were identified. Both sites exhibited positive cooperativity.
Adding myristic, stearic, or oleic acids at equimolar amounts reduced the
incorporation of [14C]palmitic acid into NAPE at low concentrations (10
[mu]M, high-affinity site) but not at high concentrations (50 [mu]M,
low-affinity site), indicating that the two putative sites can be
distinguished by their fatty acid preferences.
